Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis

Abstract Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI’s oncogenic pathways in vivo in a genetically tractable organism.


Introduction
Ewing sarcoma (EwS) is an exclusively human, aggressive tumor, typically arising from bone and soft tissues, that is reported to be the second most common bone malignancy in children, adolescents and young adults (1). Current management of EwS relies on a combination of cytotoxic drugs, surgery, and radiotherapy.
EwS is a paradigm for solid tumor development after a single genetic rearrangement: more than 80% of patients carry a fusion between the transactivation domain of the FET family member EWS RNA Binding Protein 1 (EWSR1) gene and the DNA binding domain of the E26 transformation specific (ETS) domain transcription factor Friend leukemia integration 1 (FLI1). Fusions of EWSR1 exon 7 to either FLI1 exon 6 (EWSR1-FLI1 type 1) or exon 5 (EWSR1-FLI1 type 2), account for 60% and 25% of Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusions, respectively (2). In more than 25% of EwS patients, the EWSR1-FLI1 fusion is the only detectable genetic event at diagnosis (3).
EWS-FLI is a pleiotropic oncoprotein with diverse neomorphic functions that subvert cell physiology at different levels. EWS-FLI reprograms gene expression by direct interaction with two types of DNA sequences: microsatellites made of GGAA repeats (GGAAμSats) and enhancers containing ETS consensus sequences. Upon binding to GGAAμSats, EWS-FLI acts as a pioneer factor that recruits chromatin remodeling proteins and transforms these normally quiescent genomic regions into neoenhancers that trigger transcription of hundreds of otherwise silent genes (4)(5)(6)(7). Upon binding to ETS consensus sequences, EWS-FLI displaces wild-type ETS transcription factors, hence dys-regulating the expression of their target genes (7,8). In addition, through its interaction with core subunits of the spliceosome EWS-FLI changes the ratio of protein variants produced by alternative splicing (9). These neomorphic functions are at the core of EWS-FLI's tumorigenic effect and are therefore well suited for targeted therapy.
There are many patient-derived EwS cell lines as well as human and mice cells engineered to express EWS-FLI, some of which have been used to develop xenograft and allograft models in mice and fish [reviewed in ref. (10)]. However, reproducing EwS in genetically modified transgenic animals has proven difficult. In mice, EWS-FLI expression driven by the Prx1 promoter results in abnormal muscle, cartilage, and bone development, but no tumors (11), while EWS-FLI expression driven from Osterix or Mx1 results in erythroid/myeloid leukemias, but not sarcomas (12,13). In zebrafish, a first model based on transposon-mediated expression of EWS-FLI presented malignant peripheral nerve sheath tumors, and less frequently diffuse leukemia-like and small-round-bluecell tumors (SRBCTs), but only in p53 deficient embryos (14). Recently, a new model based on Cre-inducible expression of ubi: GFP-2A-EWS-FLI in wild type zebrafish has been reported that causes rapid onset at high penetrance of SRBCTs that express canonical EWS-FLI target genes (15). Interestingly, there seems to be no spontaneous occurrence of EwS in mice or any other species, but humans (12,16,17).
Prompted by the need for genetically tractable experimental in-vivo models of the disease, we decided to investigate the effect of EWS-FLI in Drosophila. Anatomical and physiological differences between humans and Drosophila preclude the development of a realistic genetically modified model of EwS-like tumors in flies. However, given the remarkable conservation of molecular mechanisms and genes between Drosophila and humans (18)(19)(20), we hypothesized that the human EWS-FLI protein might recapitulate in flies at least some of the neomorphic functions that fuel EwS. If so, genetically modified Drosophila strains could provide a much needed genetically tractable experimental model to investigate EWS-FLI function.

Results and discussion
The human EWS-FLI fusion protein has a strong lethal effect in Drosophila As a first step towards developing a Drosophila model capable of recapitulating the oncogenic functions of the human EWS-FLI fusion protein, we tried to generate transgenic fly strains carrying the coding sequences for EWS-FLI Type 1 and Type 2 (i.e. EWS-FLI 1 and EWS-FLI 2 ) under the control of the yeast Upstream Activating Sequence (UAS). We also generated strains carrying a similar fusion between Cabeza (Caz) and Ets65A, the Drosophila orthologs of EWSR1 and FLI, respectively (Fig. 1A).
Unlike other transgenic lines that were generated at the standard rate (i.e. > 5 per 200 injected embryos), no EWS-FLI 1 nor EWS-FLI 2 transgenic flies were recovered from 800 injected embryos (400 embryos each) (Fig. 1). These results strongly suggested that both Type 1 and Type 2 EWS-FLI fusion constructs must be toxic even at the exceedingly low rate of expression that UAS transgenes can be expected to leak in the absence of Gal4. Such toxicity levels have only been observed in UAS constructs encoding very potent cell lethal proteins like Ricin A or Diphtheria Toxin A [reviewed in ref. (21)].
To circumvent this problem, we carried out a second round of injection using different landing sites and at low temperature (18 • C) to minimize leakiness. A total of 1200 embryos were injected. Once more, we failed to recover any UAS-EWS-FLI 2 transgenic flies, but we did recover one fly from embryos injected with the UAS-EWS-FLI 1 transgene (Fig. 1A). Notably, despite the difficulty of generating this fly, its offspring was fully viable and fertile and a stable line carrying the transgene was easily made.
DNA sequencing revealed that the transgene present in this line had a deletion of 17 nucleotides that causes the loss of six amino acids and a frameshift. The resulting protein is identical to EWS-FLI from amino acids 1 to 406 but lacks the 69 amino acid Cterminal tail of EWS-FLI that is replaced by a new 64 amino acid sequence. The entire EWSR1 portion as well as most of the FLI portion of EWS-FLI, including its entire DNA binding domain, remain, therefore, unaffected in this mutant that we have named EWS-FLI 1FS (Fig. 1A).
These results suggest that the strong toxicity of EWS-FLI transgenes in Drosophila maps to the 69 amino acid C-terminal tail that is lost in EWS-FLI 1FS . To test this possibility, we first investigated the viability of full-length hFLI1 transgenes, which indeed carry the same C-terminal tail. We found that full-length hFLI1 transgenes can be recovered at the standard rate (>5 per 200 embryos) thus showing that the 69 amino acid C-terminal tail is not necessarily toxic in flies (Fig. 1). We then quantified the rate of recovery of transgenic lines carrying two different mutant versions of EWS-FLI1 1 and EWS-FLI 2 . The first had the same genetic lesion found in EWS-FLI 1FS . The second had a deletion of the 69 C-terminal amino acids. No transgenic flies carrying this deletion were recovered from a total of 800 injected embryos (Fig. 1, EWS-FLI 1 69C and EWS-FLI 2 69C ). However, both EWS-FLI 1FS and EWS-FLI 2FS transgenic lines carrying the frame shift version were recovered at the standard rate (Fig. 1A, EWS-FLI 1FS and EWS-FLI 2FS ). These results demonstrate that the lower toxicity of EWS-FLI 1FS is caused by replacing the 69 amino acid C-terminal peptide by the one that results from the EWS-FLI 1FS frameshift (Fig. 1A).
The same results were obtained regarding similar protein fusions that are less frequent but equally pathognomonic of EwS like EWS-ERG, EWS-FEV, and FUS-ERG in which the EWSR1 or FLI1 portions are substituted by the corresponding sequences of other members of the FET protein family like FUS RNA binding protein or other ETS transcription factors like ERG and FEV. The full-length and the corresponding C-terminal deletion versions of these fusion proteins appear to be as toxic as EWS-FLI, and only the frameshift versions can be recovered as transgenes (Fig. S1).

The EWS-FLI 1FS variant retains neomorphic functions of full-length EWS-FLI in human cells
Work on NIH3T3 mouse fibroblasts suggested a role for the Cterminal region of FLI in mediating transcriptional down-regulation by EWS-FLI (22). However, later functional studies on the effect of an 81 C-terminal EWS-FLI deletion ruled out any significant role for this region in oncogenic transformation in an EwS cellular context (23). These results strongly suggest that EWS-FLI 1FS , which is only lacking the 69 C-terminal amino acids might also retain the neomorphic functions of full-length EWS-FLI. To test this hypothesis, we tested EWS-FLI 1FS ' effect on both gene expression and clonogenicity in human HEK293 cells. To determine the effect on gene expression, we selected a set of validated EWS-FLI target genes: FCGRT, NR0B1, NKX2-2, PPP1R1A, and SOX2 (4,(24)(25)(26). We found that like full-length EWS-FLI, EWS-FLI 1FS significantly upregulates NR0B1, PPP1R1A, and FCGRT (Fig. 1B). Neither EWS-FLI 1FS nor full-length EWS-FLI had a significant effect on the expression of NKX2-2 and SOX2. We do not know why wild-type EWS-FLI fails to upregulate NKX2-2 and SOX2 in HEK293, but it may reflect the fact that out of the five selected genes these are the two whose transcription start sites are furthest away for the GGAA repeats (63 Kb for NKX2-2 and 470 Kb for SOX2). It is thus possible that transcriptional rewiring of these genes from such distant enhancers may take longer than 72 h post transfection with EWS-FLI in HEK293 cells.
These results show that EWS-FLI 1FS retains full-length EWS-FLI's neomorphic capability to upregulate three Ewing sarcoma signature genes in HEK293 cells. We also found EWS-FLI 1FS and full-length EWS-FLI to be equally competent at significantly increasing clonogenicity in HEK293T cells (Fig. 1C). Altogether, these results are consistent with the hypothesis that EWS-FLI 1FS retains the neomorphic functions of full-length EWS-FLI.

EWS-FLI 1FS brings about a massive rewiring of the transcriptome in Drosophila
EWS-FLI brings about a major dysregulation of transcription in human cells. To determine if EWS-FLI 1FS may have similar effects in Drosophila, we undertook a genome-wide transcriptome analysis using Affymetrix. To identify a suitable cell type, we expressed EWS-FLI 1FS from a variety of Gal4 drivers. We found that EWS-FLI 1FS expression from most of these Gal4 drivers had strong deleterious effects and caused embryonic lethality (Table S1), thus showing that the EWS-FLI 1FS variant remains highly toxic. Indeed, leaky expression (in the absence of any Gal4 driver) of two copies of the UAS-EWS-FLI 1FS transgene is also lethal. These results are consistent with previous reports showing that EWS-FLI is not permissive in most cell types in human, mice, and fish (27,28).
One of the drivers that allowed for larval development was P{GawB}nubbin-AC-62 (henceforth referred to as nub-Gal4). Although generally used for its prominent expression in the wing pouch of the wing imaginal disc (29), nub-Gal4 also drives Gal4 expression in the salivary glands.
Remarkably, expression of EWS-FLI 1FS from the nub-Gal4 driver in the wing disc epithelia results in tumors that present immortal malignant neoplastic growth in allograft assays (Fig. S2). These data strongly substantiate the tumorigenic potential of the EWS-FLI fusion by itself, without the concourse of other genetic lesions. Importantly, however, these epithelia-derived tumors bear little resemblance to EwS. We, therefore, worried that tumor typespecific changes in their transcriptome could overshadow those specifically induced by EWS-FLI, which are the ones we wished to identify. To circumvent this problem, we focused our attention on EWS-FLI expressing salivary glands that appear to develop fairly normally.
During larval development, Drosophila salivary glands' secretory cells undergo multiple rounds of DNA synthesis without mitosis (i.e. endoreduplication). The resulting so called polytene chromosomes are made of hundreds of chromatids aligned to one another along their entire length and display a reproducible pattern of bands and interbands that correlate tightly with chromosome structure and transcriptional activity [reviewed in ref. (30)]. Immunofluorescence with an anti-EWSR1 antibody on nub >EWS-FLI 1FS salivary glands showed that EWS-FLI 1FS binds to polytene chromosomes in a banding pattern that largely overlaps DAPI interbands and loosely condensed bands ( Fig. 2A), which correspond to transcriptionally active genes (30).
Affymetrix profiling showed a massive change of the salivary glands' transcriptome upon nub-Gal4-driven expression of EWS-FLI 1FS with a total of 1021 and 488 genes significantly up (FC > 2) and downregulated (FC< -2), respectively (Fig. 2B, red and green dots; Table S2). In some cases, the extent of dysregulation is extreme like the 86 genes that have little or no expression in control salivary glands and are upregulated between 10 and 300-fold upon EWS-FLI 1FS expression. Notably, a third (n = 32) of these genes are associated with neural development (Fig. 2B, blue dots; Fig.  S3). Upregulation of neural genes is a common trait of EwS cells and EWS-FLI has been shown to promote an Ewing-specific neuroectodermal gene expression program in a wide variety of cell types (31)(32)(33)(34). To validate the Affymetrix data, the expression levels of nine up and eight downregulated genes in EWS-FLI 1FS and control salivary glands were confirmed by RT-qPCR ( Fig. S4 and Table S3).
We then compared our results with published transcriptomic signatures derived from two meta-analyses of dozens of EwS's cellular models and tumor samples (35,36). To this end, we identified enriched KEGG pathways and carried out gene set enrichment analysis (GSEA) (37,38). We found eight KEGG pathways that are significantly enriched (P < 0.05) in our EWS-FLI salivary gland signature, 18  Other orthologs of known targets of EWS-FLI in EwS cells like LOX, FOXO1, GLI1, or NKX2.2 were not dysregulated in our samples. However, differences in EWS-FLI-driven gene expression dysregulation are notorious even among datasets derived from human cells as substantiated by the partial overlap between different transcriptomics studies (35,36 (35,36). This is likely to result from the fact that upregulation can affect any gene regardless of the cell type in which it is normally expressed, while only genes that are expressed in the cell type under study can be downregulated (35). Downregulation of the existing cell differentiation program is a common trait of cellular EwS models (31,32). Consistently, a fifth (21/101) of the most downregulated genes (FC< -5) in our Drosophila dataset are salivary gland-specific genes.
These results reveal that expression of EWS-FLI 1FS in Drosophila brings about a major dysregulation of gene tran-

The EWS-FLI 1FS fusion interacts with Drosophila subunits of RNApol II, chromatin remodeling complexes and the spliceosome
EWS-FLI's oncogenic functions depend upon its direct interaction with specific proteins. To identify the interactome of EWS-FLI 1FS in Drosophila, we carried out co-immunoprecipitation (co-IP) with an anti-EWSR1 antibody followed by mass spectrometry (MS). We identified 631 proteins significantly enriched (FC > 3 and BFDR < 0.02) in nub > EWS-FLI 1FS salivary gland cells compared to control samples. A total of 594 of these proteins map to human orthologs. Top ranking in terms enrichment are some of the core components of transcription and ATP-dependent chromatin remodeling complexes that are known to interact with, and be required for, the oncogenic function of EWS-FLI in human cells (Fig. 3A).
Polr2A, Polr2B, Polr2C, Pol2rG, and Polr2I are subunits of the RNA polymerase holoenzyme II (RNA pol II), the synthetic apparatus of mRNA. The human orthologue of dPol2rG, RBP7, is one of the first and best characterized EWS-FLI interacting proteins in EwS cells. Pol II binding to EWS-FLI is a key step in the transcriptional upregulation of EwS signature genes (40)(41)(42). Brahma (Brm) and Moira (Mor) are core components of the Drosophila Brahma associated proteins (BAP) complex and orthologs of the equivalent human BRG1/BRM-associated factor (BAF) complex components SMARCA4 (a.k.a BRG1) and SMARCC1/SMARCC2. These, and other BAF complex proteins, have been shown to co-purify with EWS-FLI in human cells (9,43) and their recruitment to tumor-specific enhancers is a key triggering event in EwS oncogenesis (44). Mi-2 and MEP-1 are components of the Drosophila nucleosome remodeling and deacetylase (NurD) complex. Mi-2 is the ortholog of human CHD3, CHD4, and CHD5 that are structural components of the human NurD complex, which has been shown to mediate transcriptional repression in EwS (45). Human NurD complex proteins CHD4, HDAC2, and RBBP7 co-purify with EWS-FLI from EwS cell extracts (9). Imitation SWI (Iswi), the orthologue of human SMARCA1 and SMARCA5, is the core component of the Drosophila ISWI family complexes (46,47). There are no published data linking ISWI with EwS development, but SMARCA5, and four other proteins of human ISWI have been shown to co-purify with EWS-FLI from human cells extracts (9).
To further investigate the resemblance between the EWS-FLI 1FS Drosophila interactome with that of EWS-FLI in human cells, we identified enriched KEGG pathways and gene ontologies in our dataset and in two published studies of the human EWS-FLI interactome (9,50). Remarkably, in all three datasets more than half of the proteins assigned to KEGG pathways belong to one of only four KEGG categories: "ribosome," "spliceosome," "RNA transport," and "protein processing in ER" (Fig. 3B) (Fig. 3C). Five of these were also found in the Elzi et al., study. These results demonstrate that EWS-FLI 1FS is capable of interacting with the Drosophila orthologues of proteins with which EWS-FLI interacts in EwS cells, including subunits of RNApol II, chromatin remodeling complexes and the spliceosome that are essential for EWS-FLI tumorigenic effect (44,45).

EWS-FLI 1FS modulates splicing in Drosophila
Alternative splicing (AS) increases the functional diversity of proteins and noncoding RNAs and is key in the regulation of many cellular processes (51). In human cells, EWS-FLI modulates AS through its interaction with essential splicing factors like PRPF6, PRPF8, PRPF9, DDX5, SF1, and U1C (9,52). Conversely, some splicing factors have been shown to modulate the transcriptional activity of EWS-FLI (53). Prompted by the abundance of spliceosome subunits in the EWS-FLI 1FS interactome, including core subunits of the pre-mRNA splicing complex like Prp8 and Prp19 (54,55), we decided to investigate if EWS-FLI 1FS might also modulate splicing in Drosophila. To this end, we generated RNA-seq data and quantified differences in AS profiles between EWS-FLI 1FS -expressing and control salivary glands using vast-tools (56,57).
We found 273 alternative events with a PSI (delta Percent Spliced In) ≥ 15 between EWS-FLI 1FS expressing and control salivary glands. A study in EwS cell line TC32 identified 386 AS events dependent upon EWS-FLI. Taking into account that there are on average 4 introns per gene in Drosophila (Flybase, FB2021_04 Release Notes) and 8 to 9 in human (58), the number of AS events caused by EWS-FLI 1FS that we have found in Drosophila is in relative terms similar to that reported in the TC32 cell line (9).
EWS-FLI 1FS -driven AS events in Drosophila salivary glands included cassette exons, intron retention, and alternative 3' and 5' splice sites (Fig. 3D). Interestingly, among regulated introns, EWS-FLI 1FS -driven enhanced removal (n = 59) outnumbered enhanced retention (n = 21), hence suggesting that EWS-FLI 1FS activity can be rate limiting for removal of certain introns (Fig. 3D and E). On the other hand, EWS-FLI 1FS induced skipping of 78 cassette exons, while promoted inclusion of 56, indicating that EWS-FLI 1FS can act either as an activator or as a repressor of alternatively spliced exons (Fig. 3D and E). Similar effects were observed in EwS TC32 cells in which retained introns almost doubled and skipped exons dropped upon EWS-FLI depletion (9). The results derived from the bioinformatics analysis using vast-tools were validated by RT-PCR for all five genes chosen for this purpose (Fig. 3F and  S5). Sashimi plots of these splicing events are shown in Fig. S6. Included among them are mor and E(z) whose orthologs are also alternatively spliced in human EwS TC32 cells (9).
These results show that, as in EwS cells, EWS-FLI 1FS interaction with the spliceosome modulates splicing in Drosophila.

EWS-FLI 1FS dysregulates ETS domain transcription factors' targets
EWS-FLI interferes with enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors, hence disrupting the expression of ETS target genes (7). To assess the effect of EWS-FLI 1FS expression on ETS target genes in Drosophila salivary glands, we ran the EWS-FLI 1FS transcriptomic signature (Table S2) against the TF2DNA 2018 database of transcription factor binding motifs and regulated genes using FlyEnrichr (59,60). We found that all eight ETS domain transcription factors of Drosophila score very high, with adjusted P-values < 1.0E−10 and combined scores between 20 and 110 (Fig. 4A).
One of the ETS domain transcription factors that is most significantly affected is E74, which is encoded by the Eip74EF gene (adj-P value = 9.4E−20; combined score = 71). E74 is essential for the regulation of many ecdysone secondary-response genes. In salivary glands, there are eight and three known targets genes for which E74 is a positive and negative regulator, respectively (61,62). Remarkably, most of these genes are strongly dysregulated upon EWS-FLI 1FS expression (Fig. 4B). These results underpin the complexity of the mechanisms through which EWS-FLI rewires the transcriptome and suggest that, as in human cells, EWS-FLI is able to outcompete natural ETS transcription factors in Drosophila (7).

EWS-FLI 1FS drives BAP-dependent transcription from GGAAμSat sequences
BAP complex-dependent conversion of silent GGAAμSats into upstream activating sequences or neo-enhancers accounts for a significant fraction of the genes that are upregulated by EWS-FLI in EwS. GGAAμSats of around 20 GGAA repeats are best suited as EWS-FLI responsive elements (4,5,7,44,63).
Our results showing that EWS-FLI 1FS co-immunoprecipitates with subunits of RNApol Il and chromatin remodeling complexes, including BAP/BAF itself, suggest that this fusion protein may also be capable of driving transcription from GGAAμSats in Drosophila. However, in stark contrast with the human genome that contains thousands of GGAAμSats in the range between 18 and 22 GGAA repeats in length (64), GGAAμSats are extremely rare and much shorter in Drosophila: the r6.40 reference genome contains only 83 GGAAμSats longer than 2 GGAA consecutive repeats and the longest one in the entire genome has only 9 GGAA repeats.
To make it possible, to test if the EWS-FLI 1FS fusion can trigger the chain of events that activate transcription from GGAAμSats in Drosophila, we generated a new set of genetically engineered fly models carrying GGAAμSats that had 1, 3, 10, and 20 GGAA repeats upstream the heat shock minimal promoter followed by the YFP coding sequence (Fig. 4C). For technical reasons, these experiments were carried out driving EWS-FLI 1FS transcription from sal EPv -Gal4, that like nub-Gal4 is also expressed in the salivary glands. We observed no YFP signal upon EWS-FLI 1FS expression in salivary glands carrying 1x(GGAA)μSat > YFP or 3x(GGAA)μSat > YFP transgenes. However, YFP fluorescence levels were high, around 100 and 150-fold over background level, in salivary glands expressing EWS-FLI 1FS and carrying 10x(GGAA)μSat > YFP and 20x(GGAA)μSat > YFP transgenes, respectively (Fig. 4D). EWS-FLI 1FS can also upregulate YFP expression from the 20x(GGAA)μSat > YFP transgene in other tissues like wing discs (sal EPv -Gal4), eye discs (GMR-Gal4), and the neuroepithelium (ogre-Gal4) (Fig. S7). In all these cases, development of the EWS-FLI 1FS -expressing tissue is severely compromised.
Notably, the type 2 fusion EWS-FLI 2FS is equally capable of activating YFP transcription from the 20x(GGAA)μSat, and notably, so is the Caz-Ets65A fusion made between the Drosophila homologues of human EWSR1 (Caz) and FLI1 (Ets65A) although with a lower productivity than its human counterpart (Fig. S8).
To determine if as in EwS cells, EWS-FLI 1FS -driven transcription from GGAAμSats requires the BAF chromatin remodeling complex in Drosophila, we quantified YFP fluorescence in salivary glands that in addition to carrying the 20x(GGAA)μSat > YFP transgene and expressing EWS-FLI 1FS were depleted for each of ten members of the BAP/BAF complex by RNAi. We found that depletion of either mor (Hs SMARCC1 and SMARCC2) or Snr1 (Hs SMARCB1) essentially eliminates EWS-FLI-dependent YFP expression from the 20x(GGAA)μSat, which is also reduced to a greater or lesser extent by depletion of any of the ten components of the complex that we tested ( Fig. 4D and S9).
These results demonstrate that EWS-FLI 1FS can productively interact with and convert transcriptionally silent GGAAμSats into neo-enhancers to bring about BAF/BAP-dependent unscheduled transcription in Drosophila cells. In addition, these results further substantiate the conclusion that the C-terminal region affected in EWS-FLI 1FS is not essential for driving transcription from GGAAμSats.

The BAP/BAF complex is required for GGAAμSat-independent EWS-FLI 1FS -driven dysregulation of the Drosophila transcriptome
Having found that BAF/BAP is essential for EWS-FLI-driven transcription from GGAAμSats we asked whether this chromatin remodeling complex might also be required for the massive rewiring of the transcriptome generated by EWS-FLI 1FS in salivary glands, which indeed cannot be accounted for by the few and short GGAAμSats that are present in the Drosophila genome.
To answer this question, we investigated the effect of loss of mor on the EWS-FLI 1FS -dependent transcriptome. We found that nearly three quarters (73%, n = 596) of 816 genes that are upregulated in salivary glands expressing EWS-FLI 1FS from the sal EPv -Gal4 driver are not significantly upregulated in salivary glands that in addition to EWS-FLI 1FS express mor-RNAi (Fig. 4E and F). Likewise, over half (60%, n = 142) of 237 genes that are downregulated by EWS-FLI 1FS are not in EWS-FLI 1FS expressing salivary glands that are depleted of mor ( Fig. 4E and G). These results show that a large fraction of the EWS-FLI 1FS signature is dependent upon the Drosophila orthologue of human BAF HsSMARCC1 and SMARCC2 subunits. Consistent with these results, heat maps show EWS-FLI 1FS expressing samples to be clearly distinct from mor-depleted, EWS-FLI 1FS expressing samples both for genes upregulated and downregulated by EWS-FLI 1FS (Fig. 4H and I). However, hierarchical clustering shows mor-depleted EWS-FLI 1FS expressing samples to be closer to control for genes upregulated by EWS-FLI 1FS , but closer to EWS-FLI 1FS expressing samples for downregulated genes ( Fig. 4H and I, and Fig. S9B and C). These results strongly suggest that the BAP/BAF chromatin remodeling complex is necessary for the dysregulation of more than half of the EWS-FLI 1FS Drosophila signature, which, indeed, is GGAAμSat independent.
Altogether, we have found that the human EWS-FLI, EWS-ERG, EWS-FEV, and FUS-ERG fusion proteins are so strongly toxic to Drosophila that we could no generate lines carrying UAS transgenes encoding any of these oncogenes. However, we have identified a spontaneous mutant frameshift variant that circumvents this limitation thanks to the new C-terminal 64 amino acid tail.
Remarkably, human EWS-FLI 1FS can interact with the fly homologues of many of the EWS-FLI human protein partners that are core subunits of chromatin remodeling complexes, transcription machinery, and spliceosome. Through these interactions, EWS-FLI 1FS is able to recreate in flies the neomorphic functions that account for the EWS-FLI oncogenic effect in humans: a massive reshaping of the transcriptome affecting, among others, targets of all eight of the fly's ETS transcription factors; de novo creation of enhancers at GGAAμSats; and changes of the ratio of protein variants produced by alternative splicing. In addition, EWS-FLI 1FS induces malignant tumors in the wing disc that, although epithelia-derived and different to EwS, are remarkable because they strongly substantiate the conclusion that EWS-FLI alone can trigger malignant growth in a living animal.
Anatomical and physiological differences preclude the generation in flies of tumors that reproduce the complexity of traits that define specific human cancer types. This is even more so in the case of EwS that stands out as the paradigm of cancer type that develops only in humans and for which even rodents have failed to provide realistic models. However, our work showing that specific neomorphic functions of EWS-FLI can be realistically reproduced in genetically engineered fly models opens up an unprecedented opportunity to investigate EWS-FLI function in vivo in this genetically tractable organism. Indeed, Drosophila has a solid track record as a model system and has made seminal contributions to our understanding of fundamental biology processes, including splicing, transcription, and chromatin remodeling. The high-content sophisticated functional assays that are standard in Drosophila research may help to unveil new paths of intervention to fight the deadly human disease.

Materials and methods
Detailed materials and methods, including fly stocks, genotypes, crossing schemes, allograft assays, immunohistochemistry, cloning and generation of transgenic lines are provided in the Supplementary Material Appendix. Microarray processing was performed using the Drosophila Genome 2.0 protocol (Affymetrix/ThermoFisher Scientific), and scanned with a GeneChip Scanner GCS3000 (Affymetrix/ThermoFisher Scientific). Alternative splicing was quantified from RNA sequencing data ob-tained from salivary glands using the toolset vast-tools v2.5.1 (56) [dm6, VASTDB library: vastdb.dme.23.06.20.tar.gz (57)]. Samples for nano LC-MS/MS mass spectrometry were processed in an Orbitrap Fusion Lumos™ Tribrid. Detailed and fully referenced information on all these methods can be found in the Supplementary Material Appendix.